Our aims are to clarify the molecular mechanism of regulation of vascular smooth muscle contraction. It is known that an increase in the intracellular concentration of Ca2+, in the range of 10-6 M induces contraction of smooth muscle. However, the mechanism(s) by which the change in Ca2+ is detected by the contractile apparatus and then processed to result in either contraction or relaxation may be different from that of skeletal muscle where troponin-tropomyosin is known to play an important role. The most popular hypothesis to account for the regulation of smooth muscle contraction is that phosphorylation of the 20,000 dalton light chains of myosin by a myosin light chain kinase (MLCK) activates the contractile appartus and leads to contraction; relaxation is achieved by dephosphorylation of myosin by a myosin light chain phosphatase (MLCP). However, how the phosphorylation of myosin regulates the ATPase activity of actomyosin (contraction and relaxation) has not been elucidated, and the focus of this proposal is to examine the molecular mechanism of the regulation of ATPase activity of smooth muscle actomyosin. The smooth muscle myosin molecule shows two distinct conformations, a looped structure and an extended structure. We have worked on the relationships between the conformation and myosin, and phosphorylation and ATPase activity. This proposal will examine in detail different aspects of these relationships to clarify the molecular mechanism of regulation. It is hard to imagine that the large change in the shape of myosin can occur in the fiber, and it is thought that a smaller conformational change is the determinant of ATPase activity. We propose that the head-neck region of the molecule may be important in this regard. Subfragment-1 and heavy mero-myosin (HMM) are good tools to elucidate this problem. Recently, it was reported that myosin could be phosphorylated by the Ca2+ activated, phospholipid dependent protein kinase (protein kinase C). The effects of this phosphorylation on ATPase activity, conformation of myosin and thick filament formation will also be tested. Recently, we found that MLCK phosphorylates the 20,000 dalton light chain of myosin at a second site. The effect of this phosphorylation on the biological function of myosin and its conformation will be elucidated. The localization of these phosphorylation sites will also be identified by separating and isolating the phosphopeptides. These experiments should allow a more detailed assessment of the role of myosin phosphorylation and thus should aid in our understanding of the molecular mechanism of regulation in smooth muscle.